The initial year of the project Characterization of hematopoietic stem cell (HSC) and progenitor cell aging, identification of candidate age-regulated genes as targets for therapeutic intervention has been focused on establishing a solid platform for which all experiments will be built from. The Epigenetics and Stem Cell (ESC) Unit largely uses the murine model system to study the hematopoietic stem cell (HSC) compartment and the alterations that occur during aging. As such, it was critical to establish the Units animal study proposals. Once approved, the ESC unit began breeding and aging murine colonies for future experiments. Additionally, we have validated and optimized conditions for many of the fundamental experiments we use for the study of HSCs. We have established a standard operating procedure for myleo-ablative murine transplant conditions together with the Comparative Medicine Section by establishing lethal irradiation conditions for amenities here, and optimized injection procedures that will allow for hematopoietic stem cell (HSC) transplants to study the functional potential of the cells. We have also worked towards re-establishing working protocols for the ESC Units RNA isolation, DNA damage analysis, cell culture techniques, immune-histochemistry, and FACS purification at the NIA. This is central to our research progress as the design of many of the required experiments in the laboratory revolve around these techniques. We, together with the Flow Cytometry Unit, FACs purified hematopoietic stem cells from aged (21 month old) and young (3 month old) C57BL/6 male mice. We isolated two sub-populations of HSCs, which we have previously defined as myeloid-biased HSCs and lineage balanced HSCs. Cells from 15 young and 5 old mice (pooled 3 young mice for adequate cell numbers) for a total of 20 samples- 5 young CD150high, 5 CD150low, 5 old CD150high, and 5 old CD150low. Towards addressing the effect of CR on the epigenetic landscapes, we have continued to calorically restricted (CR) C57Bl/6 male mice acquired from the NIA aged CR rodent colony to 24 months of age. Bone marrow analysis and HSC purification from these cells has been performed. We treated young and aged C57BL/6 mice with 1mg/mL of the antioxidant N-Acetylcystine delivered via drinking water in 7 cohorts that included 3 young mice and 1 old mouse per group. 7 age-matched control cohorts were also included in the experiment. After exposure to the antioxidant for one month, the treated and untreated young and old age-matched mice were sacrificed. Bone marrow analysis was performed after the administration of this antioxidant for a four-week period. Two independent competitive transplant experiments were performed to establish if NAC treatment improved functional potential of young and aged HSCs. FLARE comet assays, that measure oxidative damage, are being performed on HSCs purified from treated and untreated mice.